Method for detecting potato virus and viroid by once composite PCR reaction

A potato virus and potato technology, which is applied in the field of plant virus molecular detection and plant virus detection, can solve the problems of inconvenient long-distance and large-scale sampling, inability to directly detect seed potatoes, and inability to detect viruses, etc., achieving wide practicability, low cost, The effect of easy sampling

Inactive Publication Date: 2008-01-02
HUAZHONG AGRI UNIV
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Problems solved by technology

At present, ELISA technology is used in the quality inspection of seed potatoes to detect these viruses. The main steps are: firstly, different antiserum must be prepared according to the virus. Compared with the synthesis of PCR primers, the preparation of antiserum has a complicated process and high cost; secondly , Due to the sensitivity of ELISA detection, it is not possible to detect low-concentration viruses during the incubation period. It can only be carried out by taking plant leaves during the growing season, but not directly detecting seed potatoes (tubers). Therefore, the detection center needs to collect materials from the field during the growing season , and then take it back to the laboratory for testing, it is very inconvenient to take samples (leaves) in large quantities from a long distance

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  • Method for detecting potato virus and viroid by once composite PCR reaction
  • Method for detecting potato virus and viroid by once composite PCR reaction

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1: the establishment of the basic detection method of potato virus and viroid

[0028] Step 1: Primer Synthesis

[0029] The 5 kinds of viral primer sequences designed by the present invention (see Table 1) are submitted to "China Shanghai Sangong Bioengineering Technology Service Company" to synthesize primers, and the general synthesis amount is 10OD per primer (2500 μl of dilutable volume, for 5000- 8000 responses).

[0030] The second step: small amount of total RNA extraction from leaf samples

[0031] Propagate the test-tube plantlets containing potato PVY, PVX, PVS, PLRV, and PSTV poisonous sources in MS basic medium supplemented with 3% sucrose. After the test-tube plantlets grow for 4 weeks, take about 500 mg of leaves from each poisonous source , total RNA was extracted independently. The RNA extraction method is as follows: 500 mg of leaf samples are immediately put into liquid nitrogen and ground into powder, then placed in a 5ml centrifuge tub...

Embodiment 2

[0039] Example 2: Detection of Potato Viruses and Viroids in Potato Leaves Planted at Different Altitudes

[0040] Step 1: Primer Synthesis

[0041] The operation steps are the same as the first step of embodiment 1.

[0042] Step 2: Off-site leaf sampling

[0043] The sampling site is Enshi, Hubei, at four different altitudes (100M, 500M, 1100M, 1640M). Select plants with symptoms of the virus in the potato planting area, and use scissors to cut the expanded leaves with the top of the plant downward. The sampling volume is about 5 leaves. After each plant sample is taken, the scissors are disinfected with 75% alcohol before proceeding to the next one. sampling. Each sample was individually packed into a polyethylene food bag, and then put into a sampling box for storage and brought back to the laboratory. The ice packs in the sampling box were pre-cooled in the refrigerator the day before sampling.

[0044] Step 3: Small amount extraction of total RNA from leaf samples f...

Embodiment 3

[0053] Example 3: Detection of Potato Viruses and Viroids in Potato Tubers

[0054] Step 1: Primer Synthesis

[0055] With the first step of embodiment 1.

[0056] Step Two: Tuber Sampling

[0057] The sampling location for the detection of potato tuber virus was carried out at Huazhong Agricultural University in Wuhan City, Hubei Province, China. A total of 10 varieties were tested in this experiment, of which 5 were tubers planted in greenhouse pots and 5 were planted tubers in the experimental field. The 5 varieties planted in the greenhouse were sampled, and one tuber was taken from each pot. The sampling method of the 5 varieties planted in the experiment was that 5 plants were randomly selected for each variety, and 1 tuber was taken from each plant. After the tubers were brought back to the laboratory, the soil was first washed away with tap water, then washed twice with double distilled water, and dried at room temperature. Cut 5 tubers of each variety into small pi...

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Abstract

The invention discloses a detecting technique of potato virus and viroid molecule and atopic primer, which is characterized by the following: detecting four potato viruses (PVX, PVY, PLRV and PVS)and one potato viroid (PSTV)through once compound PCR; improving the atopy and detecting efficiency with low cost.

Description

technical field [0001] The invention belongs to the technical field of plant virus detection, in particular to the technical field of plant virus molecular detection. Utilizing PCR technology, a reverse transcription polymerase chain reaction (PCR) method (m-RT-PCR) for simultaneously detecting 5 kinds of potato (like) viruses. The invention also relates to a primer design method in m-RT-PCR reaction, and reaction system parameters and program parameters for simultaneous detection of five potato viruses. Background technique [0002] Virus detection is an important link in the production of virus-free seed potatoes, and is the quality assurance of virus-free seed potatoes. At present, antiserum-based enzyme-linked immunosorbent assay (ELASA) is mostly used for plant virus detection. When this technology is used for detection, corresponding specific antiserum must be prepared for each virus, and each virus must be detected independently, so the detection cost is high and the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/70
Inventor 谢从华柳俊聂碧华宋波涛M.佩蒙
Owner HUAZHONG AGRI UNIV
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